Leaf Extracts of Moringa oleifera Cultivated in Baghdad: Characterization and Antimicrobial Potential against Endodontic Pathogens

The use of medicinal plant preparations to clean and disinfect root canal infection is gaining popularity. The aim of this study was to evaluate the bioactive composition of leaf extracts of Moringa oleifera plants cultivated in Iraq (specifically Baghdad) and their antimicrobial activity against selected root canal pathogens for potential application in endodontic treatment. Materials and Methods. Moringa leaf extracts were prepared either through cold maceration or warm digestion techniques to perform an ethanolic or aqueous extraction, respectively. Phytochemical detection was performed before thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to measure flavonoids and phenolic compounds within both extracts. Then, their antimicrobial activities were investigated against Streptococcus mutans, Enterococcus faecalis, and Candida albicans through minimal inhibitory concentration (MIC), minimal bactericidal concentration (MBC), and agar well diffusion assay in comparison to NaOCl and Ca(OH)2. Results. Phytochemical screening showed several active ingredients but with higher expression of flavonoids and phenolic compounds. Also, different types of these compounds were detected through TLC and quantified by HPLC. MIC values for ethanolic extract against Streptococcus mutans, Enterococcus faecalis, and Candida albicans were 60, 65, and 55, respectively, while for aqueous extract, MIC values were 70, 80, and 50, respectively. Aqueous extract showed a higher inhibition zone than ethanolic extract for both Streptococcus mutans and Enterococcus faecalis with a statistically significant difference (p ≤ 0.001) for all tested materials except with NaOCl and Ca(OH)2 in Streptococcus mutans and Enterococcus faecalis, respectively. The ethanolic extract showed a higher inhibition zone against Candida albicans, with a statistically significant difference (p ≤ 0.001) for all tested materials. Conclusion. Ethanolic and aqueous extracts of Moringa oleifera leaves cultivated in Baghdad contain considerable quantities of phytochemicals, especially flavonoid and phenolic compounds, and demonstrated antimicrobial activities against selected endodontic pathogens. Therefore, Moringa leaf extracts could be suggested as an alternative antimicrobial material in endodontic treatment.


Introduction
Herbal materials are increasingly valued in dental and medical practice due to their antimicrobial, antioxidant, anti-infammatory, and biocompatibility properties [1].One of these medicinal plants is Moringa oleifera (M.oleifera) that belongs to the Moringaceae family and is commonly called horseradish or drumstick tree [2].Tis plant possesses signifcant nutritional and medicinal properties which make it a good source of glucosinolates, favonoids and phenolic acids, carotenoids, tocopherols, polyunsaturated fatty acids, minerals, and folate [3].M. oleifera is also claimed to possess antibacterial, antifungal, anti-infammatory, antioxidant, antiasthmatic, antiulcer, antidiabetic, antitumor, antipyretic, antiepileptic, diuretic, antihypertensive, cholesterol lowering, and hepatoprotective properties [4].Te biochemistry of M. oleifera varies according to the cultivation region, and such variation in the chemical composition can result in divergent efectiveness on health problems and safety related to its intake [5].
In endodontic treatment, the dominant factors for pulpal and periapical infammation are bacteria and their byproducts [6].Tus, elimination of them from the contaminated root canal system by both mechanical and chemical means is essential to achieve successful results [7].Lately, the use of herbal medicines in dental treatment has increased, giving the advantages of availability, lesser toxicity, and cost efectiveness [8].Herbal agents have been used in dentistry as anti-infammatory agents, antimicrobial plaque agents, antioxidants, analgesics, endodontic irrigants, and medicaments [9,10].With the high nutritional and medicinal value of M. oleifera, scientifc research has been directed toward this medicinal herb with antioxidant, antiinfammatory, and antimicrobial activities as a possible source of antimicrobial function in endodontic treatment [11].Leaf and seed extracts demonstrated antibacterial activity against E. faecalis in vitro and in the root canal ex vivo [12][13][14].Tus, the present study focused on the bioactive composition and biological functions of leaf extracts from M. oleifera plants cultivated in Iraq (specifcally Baghdad) to explore their antimicrobial action against selected root canal pathogens.

Plant Collection and
Classifcation.Fresh M. oleifera plant leaves were collected from the plant research garden of the Department of Biology/College of Science, University of Baghdad.Te plant was identifed and authenticated properly at the Herbarium of the College of Science, University of Baghdad.Te leaves were left to dry in the shade at room temperature [15] and then ground into a fne powder using an electric blender.

Plant Extracts Preparation.
Two types of plant extracts were prepared.Ethanolic extract was prepared by the cold maceration method as described by Ibrahim and Kebede [16].Using this technique, 100 g of dried leaf powder was added gradually to 1000 mL of 80% ethanol (PanReac AppliChem, Spain) in a glass beaker under stirring and stored for 72 hours.Te extract was fltered via Whatman flter paper No. 1 and dried by using a rotatory evaporator (Heiodolph, Germany) at 60 °C to obtain the dried extract (residue).Te residue was stored at 4 °C until use.Te aqueous extract was prepared by the digestion method described by Abubakar and Haque [17] as follows: 50 g of the dried leaf powder was added to 1500 mL of distilled water and placed on a hotplate magnetic stirrer (Rlabinco, the Netherlands) at 60 °C for one and a half hours before fltering through Whatman flter paper No. 1. Te fltrate was freezedried with a lyophilizer apparatus (Christ ALPHA 2-4 LD plus, Martin Christ Gefriertrocknungsanlagen GmbH).
Te percentage yields of both extracts were calculated using the following formula [18]: Percentage (%) yield � weight (g) of the concentrated extract weight (g) of the ground Moringa leaves x 100. (1) Also, pH was measured using a pH meter (WTW, Germany) for a solution of 5 mg of each residue in 10 mL distilled water.

Preliminary Phytochemical Detection of the Ethanolic and
Aqueous Extracts.Te following tests were then used to detect the presence of polyphenols, favonoids, tannins, alkaloids, saponins, and polysaccharides.

Test for Flavonoids. Tis was performed according to
Abubakar and Haque [17] and Shri Chengama Raju and Wing Kei [19].A few drops of sodium hydroxide solution were added to a glass tube containing 1 mL of extract, which turned the solution to an intense bright yellow color.Ten, a few drops of dilute acid were added.If this turns the solution colorless, it indicates the presence of favonoids.[17].Te potassium bismuth iodide solution (Dragendorf's reagent) was freshly prepared using 60 mg of bismuth subnitrate Bi(NO 3 ) 3 .H 2 O that was dissolved in 0.2 ml HCL (solution A) and 600 mg potassium iodide KI in 1 ml distilled water (solution B).Ten, solutions A and B were mixed together.Following that, 1 mL of this reagent was added to the extract in a glass tube.Te formation of an orange brown precipitate indicates the presence of alkaloids.

Test for Tannins.
A few drops of 1% lead acetate were added to 1 mL of the extract in a glass tube leading to the appearance of white or gelatinous precipitate.Tis indicates the presence of tannins [20,21].

Test for
Glycosides. 1 mL of extract solution was placed in a glass tube and treated with a few drops of Benedict's reagent (alkaline solution containing cupric citrate complex) before boiling in a water bath for 5 minutes and then cooling.Te formation of a reddish brown precipitate means that a reducing sugar is present [22].
A thin layer chromatography, aluminum-backed TLC (SiliCycle, Canada), was activated at 100 °C for 30 minutes in an oven and cooled at room temperature before use.All standard solutions for favonoids and phenolic compounds were prepared at a concentration of 1 mg/mL in absolute methanol, including cafeic acids CA, rutin R, catechin CAT, epicatechin Ep, chlorogenic acid Ch, hydroquinone H, gallic acid G, cinnamic acid Cin, kaempferol K, quercetin Q, quarcitrin Qa, luteolin L, paracumaric acid PC, pyrogallol P, and apigenin A. One spot from standard solutions and one from each sample (ethanolic and aqueous extracts in concentration of 5 mg/mL) were placed on a TLC plate using capillary tubes.Te plate was placed in a TLC jar containing the selected solvent.At the end of the solvent development, the developed TLC plates were air-dried and observed under ultraviolet light, UVT-260D Dual UV Transilluminator (Optima, Japan) at both 254 nm and 366 nm.Calculation of the retardation factor R f value was done according to the following formula [27]: distance traveled by the compound distance traveled by the solvent . (2) 2.5.High-Performance Liquid Chromatography (HPLC) (Quantitative and Qualitative Assessment).Both ethanolic and aqueous extracts in 10 mg/mL methanol were prepared for the detection of favonoids and phenolic compounds and analyzed using an HPLC system (Shimadzu, Japan).Te conditions for detection were as shown in Table 1 for phenolic compounds and Table 2 for favonoids.Te concentration of the detected compounds was measured according to the following formula [28]: Concentration of unknown � area of unknown area of known X concentration of known. (3) 2.6.Antimicrobial Testing.Microbial cultures used were microbial strains of Streptococcus mutans (OP198206.1),Enterococcus faecalis (OM250466.1),and Candida albicans (OP683214.1)that were previously isolated and identifed through real-time PCR.Tese microbes were selected because of their signifcance in endodontic infections [29].Te microbial strains (Streptococcus mutans, Enterococcus faecalis, and Candida albicans) were cultured in Muller Hinton broth (Oxoid Ltd., United Kingdom) and incubated overnight in an Electro-Termal Constant-Temperature Incubator (Laboao, China) at 37 °C and then diluted with 1 : 10 dilution factor with Muller Hinton broth (Oxoid Ltd., United Kingdom).Te turbidity of the suspensions was adjusted to obtain 0.5 McFarland standard, which estimates a concentration of (1 × 10 8 CFU/ml).Tese microbial suspensions were used in the following test.

Minimum Inhibitory Concentration (MIC).
Te MIC is defned as the lowest concentration of the antimicrobial agent which has the ability for complete inhibition of microorganism growth in tubes or microdilution wells as noticed by the unassisted eye [30].
Muller Hinton broth was prepared aseptically.Muller Hinton broth was used to prepare seven concentrations of each extract.Tese concentrations for the ethanolic extract were 75 mg/mL, 70 mg/mL, 65 mg/mL, 60 mg/mL, 55 mg/ mL, 50 mg/mL, and 45 mg/mL, while for the aqueous extract, they were 90 mg/mL, 80 mg/mL, 70 mg/mL, 60 mg/ mL, 50 mg/mL 40 mg/mL, and 30 mg/mL.Briefy, in each row of a fat-bottom 96-well plate, about 150 μL of each extract's dilution was dispended into 11 wells (10 replications for each concentration) and one last well was used as a blank for the color changing.While, 150 μL of Muller Hinton broth was added to each well of the last column of the plate to serve as the positive control (which would later be inoculated with microbial isolation).In each well of the last row of the plate, 200 μL of Muller Hinton broth with no tested materials was added to serve as the negative control.Ten, 50 μL of the previously prepared microbial isolate suspension was added to each well except for the blank and the negative control wells.After overnight incubation at 37 °C, 30 μL of 0.015% solution of resazurin (HiMedia, India) Te Scientifc World Journal was added to each well and they were incubated again for four hours.
A color change was assessed visually in the prepared plates.Any change in color from blue to pink-orange was recorded as positive, indicating microbial growth, while no color change (blue resazurin color remained unchanged) indicated that no microbial growth had occurred.Te blank wells were used only to check if there was any reaction between the extracts and resazurin pigments.Te MIC showed the lowest concentration of the tested extract at which no color change of the medium appeared and no microbial growth was indicated [33].

Te Minimum Bactericidal Concentration (MBC).
It is the lowest concentration of the antibacterial agents which can entirely kill the bacteria [34].It was determined to use the same method as Jang et al. [35] and Prastiyanto et al. [36], and the procedure was done by taking 10 μL from wells of the lowest 3 MIC values and one sample from the concentration below the MIC value from each plate that was used to determine the MIC against the tested microorganisms for both extracts.Ten, these suspensions were spread evenly on blood agar base plates (TM MEDIA, India).Te plates were incubated for 24 hours in order to detect any microbial colony growth.When no microbial colony growth occurred from directly plated contents of these selected wells, this value was recorded as the MBC value [32].

Microbial Sensitivity Test.
Te aim is to determine the diameter of the inhibitory concentration of the ethanolic and aqueous extracts using the agar well difusion assay as described by Prastiyanto et al. [36,37].
Five diferent plates of Mueller Hinton agar were inoculated with the previously prepared microbial isolates suspension.In each plate, 4 holes of 8 mm diameter were punched aseptically with a sterile cork borer.Two of these holes received 100 μL of the MIC concentration for either ethanolic or aqueous extracts.Te other two holes received either calcium hydroxide paste material (Meta Biomed, Korea) or 3% sodium hypochlorite solution (CLORMIX, Iraq) as comparative materials.Ten, the plates were incubated overnight at 37 °C, and the diameter of the inhibition zones was measured.
Te data were analyzed using analysis of variance ANOVA and post-hoc Bonferroni multiple comparisons at the 0.05 level.

Te Plant Extract Yields.
Te percentage yields of the leaf extract of M. oleifera for both ethanolic and aqueous extracts were 32.2% and 24.8%, respectively.Tis revealed that ethanolic extract exhibited a higher yield percentage in comparison to aqueous extract.Te pHs for both extracts were similar at 5.8 which is slightly acidic.4, and the corresponding TLC plate is shown in Figure 1.Te retardation factors of the favonoids appear in Table 5, with their TLC plate shown in Figure 2.
(i) All compounds were detected in accordance with their corresponding standards, and other unknown spots were also present in both ethanolic and aqueous extracts.

Qualitative and Quantitative Analyses Using HPLC.
After calculating the retention time, the area under the peak for the standard concentration (1.5 μg/mL) of all standard phenolic compounds was used in this study as shown in the HPLC chromatogram (Figure 3(a)).Tese phenolic compounds were tested in both ethanolic and aqueous extracts, and their chromatograms are shown in Figures 3(b) and 3(c), respectively.Te concentrations of phenolic compounds with similar peaks between the standards and extract solutions were calculated and are presented in Table 6.
Te ethanolic and aqueous extracts were rich in many phenolic compounds including catechin, ferulic acid, epicatechin, cafeic acid, vanillic acid, and chlorogenic acid.While gallic acid was present in the aqueous extract, the ethanolic extract constituents did not contain gallic acid.
After calculating the retention time, the area under the peak for standard concentrations (1 μg/mL) of all favonoids standards used in this study was as shown in the HPLC chromatogram in Figure 4(a).Tese favonoids were tested in both ethanolic and aqueous extracts, and their chromatograms are shown in Figures 4(b) and 4(c), respectively.Te concentrations of favonoids with similar peaks between the standards and extract solutions were calculated and are presented in Table 7.
Te ethanolic and aqueous extracts were rich in the following favonoids: rutin, hesperetin, apigenin, kaempferol, and coumarin.However, myricetin was not detected in either of the extracts.

MIC and MBC Values for the Ethanolic and Aqueous
Extracts against Selected Oral Pathogens.MIC and MBC mean values (n � 3) for both ethanolic and aqueous extracts against Streptococcus mutans, Enterococcus faecalis, and Candida albicans are presented in Table 8, while Figure 5 shows blood agar plates for MBC value determination after 24 hours of incubation for both ethanolic and aqueous extracts.Lower concentrations of the ethanolic extract were required for MIC and MBC of both Streptococcus mutans and Enterococcus faecalis in comparison to the aqueous extract.However, the aqueous extract required lower concentrations to obtain MIC and MBC values against Candida albicans.
3.6.Sensitivity Test.Te mean values of the measured inhibition zones in (mm) of both the ethanolic and the aqueous extracts in comparison with calcium hydroxide and sodium hypochlorite against Streptococcus mutans, Enterococcus faecalis, and Candida albicans are illustrated in Figure 6.Te aqueous extract showed a higher inhibition zone than the ethanolic extract for both Streptococcus mutans and Enterococcus faecalis (27.6 ±0 .9 and 25.6 ± 1.7, respectively).Tese values showed a statistically signifcant diference (p ≤ 0.001) for all tested materials except with NaOCl and Ca(OH) 2 in Streptococcus mutans and Enterococcus faecalis, respectively.However, the ethanolic extract had a higher inhibition zone against Candida albicans with a statistically signifcant diference (p ≤ 0.001) for all tested materials.

Discussion
In this study, phytochemical screening and qualitative and quantitative analyses of M. oleifera leaf extracts were performed and their antimicrobial activities against certain root canal pathogens were identifed.
Te leaf extraction was done using two diferent techniques and solvents: cold maceration with ethanol and digestion extraction with water.Obtaining the percentage yield of extract is a particularly signifcant aspect in phytochemical extraction to assess the efciency of the standard extraction for a particular plant, diferent parts of the same plant, or diferent solvents used [38].Te cold maceration method exhibited a higher yield of extract, which could be an efect of the extracting solvent nature, owing to the presence of various compounds with diferent chemical properties and polarities that may or may not be soluble in a particular solvent [39].Te diferences between ethanolic and aqueous extract yields may be due to the efciency variance of the extracting solvents in dissolving endogenous compounds from the plant material [40].High temperature of processing conditions, on the other hand, may lead to losing parts of the natural antioxidants from extracts, as heat may accelerate oxidation and other degenerative reactions [39].Tis could explain the decrease in the extract yield in aqueous extraction.Also, increasing drying temperature causes a degradation of phenolic compounds, with a signifcant reduction in the antioxidant activity of the extracts [41].Hence, the preferred drying condition for the plant was in the shade, which is superior to drying in an oven or in the sun [42].
Te results of the present study agree with the fnding by Vongsak et al. [43] that maceration with 70% ethanol is the most efcient pharmaceutical method for M. oleifera leaves Te Scientifc World Journal extraction, giving higher percentage yield, with the highest number of favonoids and phenolic compounds and the most potent antioxidant activity.Chigurupati et al. [44] found that Moringa leaf extraction by maceration with 70% ethanol was convenient and cost-efective and produced more yield (about 14%).However, the result disagrees with the Muhammad et al.' [45] study which showed that the aqueous extract gave a higher percentage yield than that of the ethanolic extract.Tis diference could be due to the diferent geographical conditions in the places where the plant leaves were collected [46] and polarities of diferent compounds present in the leaves [47].6 Te Scientifc World Journal Phytochemicals are various groups of naturally occurring secondary metabolites that are biosynthesized by plants and have biological importance due to their vital role in the plant defense mechanism against diferent pathogenic microbes [48].Studies that reported on the active phytochemicals of the M. oleifera plant were not uniform, and there was incompatibility in their reports.Tis is possibly due to the diferences in season and agroclimatic locations of the plants [49], genetic impacts, cultivation, drying, and the method used for extraction [50].It is preferable to identify the active ingredients when studying each medicinal plant due to the variation in the cultivation areas which could be associated with the presence of secondary metabolites of the plants in response to various environmental conditions.Such infuence was reported in a study [51] which demonstrated that the height and biomass of the plant can be reduced in cases where water is lacking in comparison to ordinary conditions of cultivation, while glucosinolate quantity may be enhanced.Other factors, such as the type of solvent used and its concentration, the ratio of liquid to solid and particle size of the plant material, pH, temperature, and time, could have a signifcant infuence on the efcacy of solvent extraction [52].Polar solvents, for example, were utilized to extract polyphenols from plants [39].
Te preliminary phytochemical analysis in this study confrmed the presence of alkaloids, favonoids, glycosides, polyphenols, saponins, and tannins in both ethanolic and aqueous extracts of the leaves, with polyphenols and favonoids being the most prominent detected compounds.Te fact that these bioactive compounds were identifed in leaves in great amounts may explain their pharmacological activity, as several in vitro and in vivo studies have confrmed antioxidants, antiinfammatory, immunomodulatory, and anticancer properties of M. oleifera [53].Tese results are in agreement with diferent previous studies that studied the phytochemical screening of ethanolic and aqueous M. oleifera extracts [26,[54][55][56].While Patel et al. [57] indicated the presence of the same compounds  Te Scientifc World Journal in both ethanolic and aqueous extracts, but tannins were detected only in the ethanolic extract and glycoside was lacking in both extracts.Phenolic acids are derived from hydroxybenzoic acid and hydroxycinnamic acid which are naturally present in plants, while favonoids are synthesized by the plant in reaction to microbial infections, with a benzo-c-pyrone ring as a common structure [58].Phenolic compounds represent the largest group of plant secondary metabolites and are valued for their anti-infammatory, antihepatotoxic, and antioxidant properties and free radical scavengers [59].Tese compounds have an inhibitory efect on microorganism growth which is proportional to the content of phenolic compounds in the plant extract [60].Terefore, the TLC and HPLC analyses focused on the detection of these compounds in both extracts.
Tin layer chromatography (TLC) is an easy, inexpensive, rapid, and commonly utilized method to analyze Te Scientifc World Journal and isolate small organic natural and synthetic products [61].In the current study, TLC analysis gave a signifcant result indicating the presence of a number of important phenolic and favonoid compounds.Te most suitable solvent system was found to be chloroform : glacial acetic acid : methanol (4 : 5 : 1), which provided the best separation of the active compounds.Tis might be due to its polarity which was able to provide solubility and balancing of the  Te Scientifc World Journal sample afnity for the solvent and the stationary phase to accomplish the separation of compounds within samples [62].Te results of the present study are in agreement with Chauhan et al. [21] who also used TLC to demonstrate the presence of cafeic acid, chlorogenic acid, gallic acid, and quercetin and obtained similar fndings to this study.In addition, Marrufo et al. [63] found that both aqueous and ethanolic extracts demonstrated the presence of catechin, epicatechin, kaempferol, and quercetin.HPLC can be applied for the separation, identifcation, and quantifcation of the compounds present in extracts such as polyphenols [64].Abd Rani et al. [65] identifed diferent favonoids and phenolic compounds which can be detected within M. oleifera leaf extracts.Hence, markers of these compounds were used in the present study.Te HPLC results revealed that both ethanolic and aqueous extracts contained various types of phenolic and favonoid compounds.HPLC analysis was performed under specifc conditions and at two specifc wavelengths: 280 nm for the phenolic compounds and 254 nm for the favonoids, specifc mobile and stationary phases.Terefore, not all chemical compounds in the ethanolic and aqueous extracts were detected.Te measured compounds were only those that separated under the HPLC conditions provided and had optimal absorbance at these wavelengths.Te diferent environmental conditions in diferent countries, i.e., in temperature, rainfall, sunlight, soil characteristics, and altitude [66], in addition to diferences in the harvesting season, plant genetics, maturity of the leaf, and the drying and extraction method may be the reasons for some diferences in the range of values reported in this study in comparison with other similar studies [53].Furthermore, variations in the polarity of solvents and their difusion strengths, the structural complexity, and selective solubility of secondary metabolites in a particular solvent may explain such variation in phenolic and favonoid contents within an extraction solvent [67].
Te results are in agreement with Karthivashan et al. [68] as they demonstrated the presence of favonoids: apigenin, kaempferol, and quercetin, in the 90% ethanolic extract of M. oleifera leaves.Another study [69] revealed the presence of chlorogenic acid, ferulic acid, gallic acid, and p-coumaric acid in both aqueous and ethanolic extracts.Also, Muzammil et al. [41] demonstrated a similar fnding but with the addition of p-coumaric acid and sinapic acid which were not detected in the current study.Tese diferences may be due to factors such as the development stage of leaves and handling at the time of harvesting, genetic variance, and diferent agroclimatic conditions [70].
However, the results showed that the aqueous extract contained a larger quantity of favonoids than those within the ethanolic extract.Tis is in agreement with diferent studies [71][72][73] which demonstrated that water acts as a strong extraction medium capable of dissolving most of the phenolic and favonoid compounds.While Nobossé et al. [67] concluded that ethanol was a more efcient solvent for extracting high favonoid content exhibiting higher antioxidant activity for M. oleifera leaf compared to aqueous extract.
For antimicrobial efect assessments, resazurin microtiter assay was selected since it is a simple, sensitive, and reliable method, which can give fast results and at considerably low cost [74,75].negative and Gram-positive bacteria [77].At the molecular level, favonoids can complex with proteins via nonspecifc forces such as hydrogen bonds, hydrophobic efects, and covalent bond formation.Consequently, these bioactive compounds could inactivate the adhesions of the microbes, enzymes, and cell envelope transport proteins and may be able to disrupt microbial membranes [78].Also, phenolic compounds hold an active hydroxyl group that permits the phenols to engage in hydrogen bonding with bacterial membranes which result in membrane disruption that can lead to inhibition of membrane transport, failure to sustain pH gradient, and improper regulation of the ATP level [79].Moreover, it was reported that antibacterial ability will increase as pH decreases [80].Te pH values of these extracts were slightly acidic, hence another cause for antimicrobial action.
S. mutans are Gram-positive facultative anaerobic bacteria, which play an important role in the development of oral bioflm through the production of extracellular polysaccharides [77].Tese bacteria have the ability to synthesize considerable quantities of glucan from dietary sucrose, providing binding sites for cariogenic bacterial colonization on the tooth surface [81].Moreover, S. mutans strains were found in infamed pulp at a high prevalence in both asymptomatic and symptomatic endodontic infections [82,83].According to the results of this study, M. oleifera leaf extracts showed antibacterial activity against S. mutans.Tis is in agreement with a previous study done by Elgamily et al. [84].Te activity of favonoids in the inhibition of S. mutans, such as catechins, may be due to complexing activities [85] and apigenin through increasing S. mutans membrane proton permeability and inhibiting bacterial acid production [86].
E. faecalis bacteria are responsible for multiple oral illnesses, such as dental abscess, apical periodontitis, and persistent endodontic infections [87], and considered the most prevalent bacteria in cases of endodontic treatment failure [10,88].Tese bacteria can form bioflms in aerobic, anaerobic, abundant, or insufcient nutrition environments [88] and can resist chemomechanical root canal preparation and are antiseptic [87].Tis study showed that the antimicrobial efects of ethanolic and aqueous extracts of M. oleifera against E. faecalis were similar to those of 3% sodium hypochlorite.Tis antibacterial activity is in agreement with previous studies [12,54].Tis is possibly due to the presence of favonoids in the extracts, namely, apigenin, rutin, and luteolin which have antibacterial activity against E. faecalis [86].
C. albicans, a fungus that can colonize the root canal's dentinal wall, penetrate the dentinal tubules, and form bioflms [89], as reported in persistent posttreatment apical periodontitis, can switch between blastospore and hyphal form and thus can invade the host tissue and avoid phagocytosis by macrophages and resist a wide range of pH and harsh environments allowing it to cause persistent infection [90].Te results of the current study also demonstrated an antifungal efect of M. oleifera leaf extracts against C. albicans which is in agreement with previous reports [91][92][93].Te antifungal activity could be explained by the ability of phenolic compounds to disrupt the homeostasis of Ca +2 and H + ions, upregulation and downregulation of gene transcription, breakdown of membrane integration, and impairment of the biosynthesis of ergosterol in C. albicans [94].Flavonoids such as luteolin, quercetin, and rutin also possess inhibition activity against C. albicans [86].However, the current results disagree with Moyo et al. [95] and Patel et al. [57] who found no antifungal activity of M. oleifera aqueous and ethanolic extracts against C. albicans.Tis diference is possibly due to the variation in the chemical composition and quantity of compounds in the extracts of the current study as a result of diferences in environments from which the plant leaves were gathered, the season, and the physiological status of the plant [95].
Diferences in the range of MICs between the results of this study and other studies may be attributed to many factors such as temperature, inoculation size, and microorganism type used [96].In addition, the properties of extracts are infuenced by several factors, such as plant components and fresh or dried varieties, which are in turn afected by climate, harvesting time, extraction method, solvent type, and stability of components [97].Another plausible reason could be the use of Iraqi-isolated bacterial species that may be more resistant to antimicrobial agents [98].In particular, the high prevalence of antimicrobial resistance in Iraq may be related to the unnecessary use of antibiotics [99].
Although this study used two common extraction methods which were easy and simple, there are other extraction protocols with a range of technologies which could have revealed the presence of diferent content within M. oleifera leaf [100].Also, this study investigated the antimicrobial function of M. oleifera leaf extracts through the in vitro well difusion method since there are diferences in the microbial resistance between planktonic and bioflms against antimicrobial agents [77].Further studies are required to investigate the antibioflm action and cytotoxicity of these extracts and each of their active components and their possible adverse reactions.

Conclusion
Ethanolic and aqueous extracts of M. oleifera leaves that were cultivated in Baghdad and evaluated in this study contained considerable quantities of medicinal phytochemicals, particularly favonoids and phenolic compounds as confrmed by TLC and HPLC tests.Tese extracts demonstrated powerful antimicrobial activities against different endodontic pathogens, including S. mutans, E. faecalis, and C. albicans, and this property can be employed in the construction of useful endodontic treatment materials in gel or solution form, as irrigation solutions or intracanal medication.Although these in vitro results were encouraging, further isolation and characterization of individual active constituents from these extracts and identifcation of their antimicrobial properties, evaluation of the extracts' anti-infammatory efect, biocompatibility, and safety must be performed in preclinical and clinical research studies to confrm the possible utilization of this plant and its derivatives for endodontic treatments.

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Figure 5 :
Figure 5: Blood agar plates for MBC value determination after 24 hours' incubation for the ethanolic and aqueous extracts against Streptococcus mutans, Enterococcus faecalis, and Candida albicans.
[17] for Polyphenols.As described by Abubakar and Haque[17], 1 mL solution of extract was mixed with 5% ferric chloride solution.Te formation of a brown precipitate indicates the presence of polyphenols.

Table 1 :
HPLC conditions for phenolic compounds.

Table 2 :
HPLC conditions for favonoids.Diferent active compounds were observed within the ethanolic and aqueous extracts as shown in Table3.Tese included polyphenols as the major constituents, in addition to favonoids, alkaloids, and tannins.Other components such as saponins and glycosides were also identifed in both extracts but in lesser amounts.

Table 3 :
Active compounds detected within ethanolic and aqueous extracts of M. oleifera.

Table 4 :
Retardation factor results for the phenolic compounds.

Table 5 :
Retardation factor results for the favonoids.

Table 6 :
HPLC results showing retention time (min), area under the curve, and the concentrations (μg/mL) of phenolic compounds for ethanolic and aqueous extracts.

Table 7 :
HPLC results showing retention time (min), area under the curve, and the concentrations (μg/mL) of favonoids for ethanolic and aqueous extracts.

Table 8 :
Values of MIC and MBC.